Project Summary: Investigating the role of autophagy in pancreatic cancer radiation resistance Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with limited therapeutic options. While the majority of patients die from metastatic disease, up to 30% succumb to local failure. Surgery can only be performed in the minority of patients. Radiotherapy can be used to control the primary tumor, but most patients will progress. Therefore, developing ways to sensitize these tumors to radiotherapy can have a transformative impact on patients. We have previously shown that inhibition of autophagy leads to metabolic dysfunction, decreased PDAC growth, and synergizes with radiotherapy. While these concepts are being tested in clinical trials, current inhibitors such as hydroxychloroquine (HCQ) do not appear to have optimal potency and specificity to durably block autophagy in patients and more robust inhibitors are in development that target the early phases of autophagy. One such target is ATG4b, a protease that is critical for LC3 lipidation and autophagosome formation. In this regard, we have developed and validated an inducible mouse model using a dominant negative (DN) allele of ATG4b (Atg4bC74A) that potently inhibits autophagy and allows us to model the effects of the ATG4b inhibitors that are now being developed. This novel resource gives us the opportunity to explore inhibiting the early steps of autophagy in PDAC and normal tissues for therapeutic efficacy as well as toxicity. There are several critical questions that this will allow us to answer; including the differences in inhibiting the early vs. the late phases of autophagy, normal tissue toxicity with potent autophagy inhibitors, and the optimal duration of inhibition for radiosensitization. We will address these questions by exploiting this novel genetically engineered model system with inducible control of autophagy in conjunction with an autochthonous model of PDAC that closely resembles the human disease, human primary PDAC cell lines, and a platform of sophisticated image-guided delivery of radiation to pancreatic tumors that we have developed. Mechanistically, this work builds on our prior studies that has shown a critical role for autophagy in PDAC metabolism, in particular, to mitigate reactive oxygen species (ROS). Ultimately, the innovative and rigorous approach of these studies will guide the effective translation of autophagy inhibition strategies to patients and yield important information regarding the role of autophagy in the radiation response. Against this backdrop, we propose the following specific aims. Aim 1. Optimizing autophagy inhibition using a novel inducible mouse model Aim 2. Utilizing autophagy inhibition as an approach to radiosensitize PDAC. Aim 3. Investigating the role of autophagy dependent metabolism in the response of PDAC to radiation.